Deficiency in both type I and type II DNA topoisomerase activities differentially affect rRNA and ribosomal protein synthesis in Schizosaccharomyces pombe

Abstract

The synthesis of rRNA and r-proteins was studied in temperature-sensitive topoisomerase mutants of the fisson yeast Schizosaccharomyces pombe. To reduce the severity of heatshock response seen in the wild type strain, slow temperature shift-up of the cultures was used to inactivate the mutant topoisomerases. It was found that the temperature shift caused a large preferential reduction of rRNA synthesis in the top1top2 double mutant. In contrast, no preferential inhibition of rRNA synthesis was observed in top1 or top2 single mutants, although some reduction in the total RNA synthesis was observed in the top2 mutant. Thus, as observed with Saccharomyces cerevisiae (Brill et al. 1987), relaxation of supercoiled DNA structures by either topoisomerase I or II appears to be essential for efficient transcription of rRNA genes. Analysis of r-protein synthesis indicated that there were small decreases in the differential synthesis rates of r-proteins after temperature shift-up in the top1top2 mutant, but the observed negative effects on r-protein synthesis was much smaller than that on rRNA synthesis, and degradation of the newly synthesized r-proteins was observed. These observations indicate the apparent lack of tight coupling between rRNA and r-protein synthesis in S. pombe under these experimental conditions.

Topoisomerases and yeast rRNA transcription: negative supercoiling stimulates initiation and topoisomerase activity is required for elongation.

“…This especially applies to the effects of mutating topoisomerase II. The studies of Brill et al (1987) and Yamagishi and Nomura (1988), as well as the work reported in this paper, show that topoisomerase II can substitute for topoisomerase I in promoting chain elongation. This suggests that significant topoisomerase II must be present in the nucleolus even though localization studies with immunoflourescent antibodies show it to be localized to the chromosomal scaffold (Earnshaw et al 1985) and not particularly enriched in the nucleolus.…”

“…Accumulation of the large rRNAs in both budding and fission yeast requires the presence of topoisomerase activity in the cell (Brill et al 1987;Yamagishi and Nomura 1988). The accepted explanation for this observation is that topoisomerase is required for efficient RNA chain elongation, and RNA polymerase I abortively terminates in its absence.…”

“…If neither relaxation process is available (or is too slow), the accumulation of positive supercoils ahead of the polymerase will eventually reach a point where elongation ceases. In yeast, either topoisomerase I or II can supply the relaxation activity for efficient rRNA transcription (Brill et al 1987;Yamagishi and Nomura 1988). When both topoisomerases are inactivated, however, rRNA accumulation is severely inhibited.…”

Positive supercoiling of DNA greatly diminishes mRNA synthesis in yeast.

“…In these top] mutants, the normal roles of DNA topoisomerase I might be fulfilled by DNA topoisomerase II, as both enzymes are capable of relaxing positively and negatively supercoiled DNA in vivo (5,10). Simultaneous inactivation of DNA topoisomerases I and II, through the use of topi top2 double-mutants, reduces rRNA synthesis by a factor of 10 and total poly(A)+ RNA synthesis by a factor of 3 (11,12). These reductions can be attributed to changes in the topology of the DNA template, but a direct link is difficult to establish because of the multiple cellular roles of the topoisomerases.…”

Switching from Prokaryotic Molecular Biology to Eukaryotic Molecular Biology

“…With the mutant strain obtained from Yanagida and the method of pulse-chase isotope labeling to measure synthesis rates of individual r-proteins, a method adapted from the one used routinely in our E. coli ribosome projects, we obtained clear results for many r-proteins analyzed; synthesis of r-proteins continued for a long time in the absence of rRNA synthesis, and overproduced r-proteins were slowly degraded (29). The simplest interpretation was the absence of feedback repression of r-protein synthesis, which was clearly different from the E. coli system.…”